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- Sárvári Horváth, Ilona, 1960, et al.
(author)
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Effects of furfural on the respiratory metabolism of Saccharomyces cerevisiae in glucose-limited chemostats
- 2003
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In: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 69:7, s. 4076-4086
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Journal article (peer-reviewed)abstract
- Effects of furfural on the aerobic metabolism of the yeast Saccharomyces cerevisiae were studied by performing chemostat experiments, and the kinetics of furfural conversion was analyzed by performing dynamic experiments. Furfural, an important inhibitor present in lignocellulosic hydrolysates, was shown to have an inhibitory effect on yeast cells growing respiratively which was much greater than the inhibitory effect previously observed for anaerobically growing yeast cells. The residual furfural concentration in the bioreactor was close to zero at all steady states obtained, and it was found that furfural was exclusively converted to furoic acid during respiratory growth. A metabolic flux analysis showed that furfural affected fluxes involved in energy metabolism. There was a 50% increase in the specific respiratory activity at the highest steady-state furfural conversion rate. Higher furfural conversion rates, obtained during pulse additions of furfural, resulted in respirofermentative metabolism, a decrease in the biomass yield, and formation of furfuryl alcohol in addition to furoic acid. Under anaerobic conditions, reduction of furfural partially replaced glycerol formation as a way to regenerate NAD+. At concentrations above the inlet concentration of furfural, which resulted in complete replacement of glycerol formation by furfuryl alcohol production, washout occurred. Similarly, when the maximum rate of oxidative conversion of furfural to furoic acid was exceeded aerobically, washout occurred. Thus, during both aerobic growth and anaerobic growth, the ability to tolerate furfural appears to be directly coupled to the ability to convert furfural to less inhibitory compounds.
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| 2. |
- Westman, Johan, 1983, et al.
(author)
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Effects of encapsulation of microorganisms on product formation during microbial fermentations
- 2012
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In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 96:6, s. 1441-1454
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Research review (peer-reviewed)abstract
- This paper reviews the latest developments in microbial products by encapsulated microorganisms in a liquid core surrounded by natural or synthetic membranes. Cells can be encapsulated in one or several steps using liquid droplet formation, pregel dissolving, coacervation, and interfacial polymerization. The use of encapsulated yeast and bacteria for fermentative production of ethanol, lactic acid, biogas, l-phenylacetylcarbinol, 1,3-propanediol, and riboflavin has been investigated. Encapsulated cells have furthermore been used for the biocatalytic conversion of chemicals. Fermentation, using encapsulated cells, offers various advantages compared to traditional cultivations, e.g., higher cell density, faster fermentation, improved tolerance of the cells to toxic media and high temperatures, and selective exclusion of toxic hydrophobic substances. However, mass transfer through the capsule membrane as well as the robustness of the capsules still challenge the utilization of encapsulated cells. The history and the current state of applying microbial encapsulation for production processes, along with the benefits and drawbacks concerning productivity and general physiology of the encapsulated cells, are discussed.
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| 3. |
- Westman, Johan, 1983, et al.
(author)
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Encapsulation-Induced Stress Helps Saccharomyces cerevisiae Resist Convertible Lignocellulose Derived Inhibitors
- 2012
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In: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 13:9, s. 11881-11894
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Journal article (peer-reviewed)abstract
- The ability of macroencapsulated Saccharomyces cerevisiae CBS8066 to withstand readily and not readily in situ convertible lignocellulose-derived inhibitors was investigated in anaerobic batch cultivations. It was shown that encapsulation increased the tolerance against readily convertible furan aldehyde inhibitors and to dilute acid spruce hydrolysate, but not to organic acid inhibitors that cannot be metabolized anaerobically. Gene expression analysis showed that the protective effect arising from the encapsulation is evident also on the transcriptome level, as the expression of the stress-related genes YAP1, ATR1 and FLR1 was induced upon encapsulation. The transcript levels were increased due to encapsulation already in the medium without added inhibitors, indicating that the cells sensed low stress level arising from the encapsulation itself. We present a model, where the stress response is induced by nutrient limitation, that this helps the cells to cope with the increased stress added by a toxic medium, and that superficial cells in the capsules degrade convertible inhibitors, alleviating the inhibition for the cells deeper in the capsule.
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| 4. |
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| 5. |
- Westman, J., et al.
(author)
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Improved sugar co-utilisation by encapsulation of a recombinant Saccharomyces cerevisiae strain in alginate-chitosan capsules
- 2014
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In: Biotechnology for Biofuels. - : BioMed Central Ltd.. - 1754-6834 .- 1754-6834. ; 7:1, s. 102-
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Journal article (peer-reviewed)abstract
- Background Two major hurdles for successful production of second-generation bioethanol are the presence of inhibitory compounds in lignocellulosic media, and the fact that Saccharomyces cerevisiae cannot naturally utilise pentoses. There are recombinant yeast strains that address both of these issues, but co-utilisation of glucose and xylose is still an issue that needs to be resolved. A non-recombinant way to increase yeast tolerance to hydrolysates is by encapsulation of the yeast. This can be explained by concentration gradients occuring in the cell pellet inside the capsule. In the current study, we hypothesised that encapsulation might also lead to improved simultaneous utilisation of hexoses and pentoses because of such sugar concentration gradients. Results In silico simulations of encapsulated yeast showed that the presence of concentration gradients of inhibitors can explain the improved inhibitor tolerance of encapsulated yeast. Simulations also showed pronounced concentration gradients of sugars, which resulted in simultaneous xylose and glucose consumption and a steady state xylose consumption rate up to 220-fold higher than that found in suspension culture. To validate the results experimentally, a xylose-utilising S. cerevisiae strain, CEN.PK XXX, was constructed and encapsulated in semi-permeable alginate-chitosan liquid core gel capsules. In defined media, encapsulation not only increased the tolerance of the yeast to inhibitors, but also promoted simultaneous utilisation of glucose and xylose. Encapsulation of the yeast resulted in consumption of at least 50% more xylose compared with suspended cells over 96-hour fermentations in medium containing both sugars. The higher consumption of xylose led to final ethanol titres that were approximately 15% higher. In an inhibitory dilute acid spruce hydrolysate, freely suspended yeast cells consumed the sugars in a sequential manner after a long lag phase, whereas no lag phase was observed for the encapsulated yeast, and glucose, mannose, galactose and xylose were utilised in parallel from the beginning of the cultivation. Conclusions Encapsulation of xylose-fermenting S. cerevisiae leads to improved simultaneous and efficient utilisation of several sugars, which are utilised sequentially by suspended cells. The greatest improvement is obtained in inhibitory media. These findings show that encapsulation is a promising option for production of second-generation bioethanol.
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| 6. |
- Westman, Johan, 1983, et al.
(author)
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Inhibitor tolerance and flocculation of a yeast strain suitable for second generation bioethanol production
- 2012
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In: Electronic Journal of Biotechnology. - : Elsevier BV. - 0717-3458. ; 15:3
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Journal article (peer-reviewed)abstract
- Background: Robust second generation bioethanol processes require microorganisms able to ferment inhibitory lignocellullosic hydrolysates. In this study, the inhibitor tolerance and flocculation characteristics of Saccharomyces cerevisiae CCUG53310 were evaluated in comparison with S. cerevisiae CBS8066. Results: The flocculating strain CCUG53310 could rapidly ferment all hexoses in dilute acid spruce hydrolysate, while CBS8066 was strongly inhibited in this medium. In synthetic inhibitory media, CCUG53310 was more tolerant to carboxylic acids and furan aldehydes, but more sensitive than CBS8066 to phenolic compounds. Despite the higher tolerance, the increase in expression of the YAP1, ATR1 and FLR1 genes, known to confer resistance to lignocellulose-derived inhibitors, was generally smaller in CCUG53310 than in CBS8066 in inhibitory media. The flocculation of CCUG53310 was linked to the expression of FLO8, FLO10 and one or more of FLO1, FLO5 or FLO9. Flocculation depended on cell wall proteins and Ca2+ ions, but was almost unaffected by other compounds and pH values typical for lignocellulosic media. Conclusions: S. cerevisiae CCUG53310 can be characterised as being very robust, with great potential for industrial fermentation of lignocellulosic hydrolysates relatively low in phenolic inhibitors.
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| 7. |
- Westman, Johan O., et al.
(author)
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Encapsulated vs. free yeast : A comparative proteomic study
- 2011
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In: International PhD course in Industrial Biotechnology for lignocellulose based processes, October 16-21, Göteborg, Sweden.
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Conference paper (other academic/artistic)abstract
- In the search for a replacement for fossil fuels, due to their depletion as well as an increased concern about our environment, 2nd generation bioethanol comes out as one of the most promising alternatives. There are challenges in several steps of lignocellulose processing – especially due to the formation of for yeast inhibitory compounds during pretreatment and hydrolysis. It has previously been shown that encapsulation of the yeast in membranes made of an alginate gel enables the yeast to survive otherwise toxic hydrolysates. The physiological changes arising from encapsulation are however largely unknown, although it has been shown that the macromolecular composition of the yeast changes during prolonged cultivation. In this study we have therefore performed a comparative proteomic study of yeast grown in capsules and in suspension in anaerobic batch cultivations.
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| 8. |
- Westman, Johan O., et al.
(author)
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Inhibitor tolerance and flocculation : Characterization of a yeast strain suitable for 2nd generation bioethanol production
- 2011
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In: Abstract book, Joint poster exhibition at the Dept Chemical and Biological Engineering, Chalmers University of Technology, and Dept Chemistry, University of Gothenburg. April 12, Göteborg, Sweden.
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Conference paper (other academic/artistic)abstract
- Robust second generation bioethanol processes require microorganisms able to obtain high yields and production rates while fermenting inhibiting hydrolysates. However, tolerance towards inhibitors like, carboxylic acids, furan aldehydes and phenolic compounds, is still an issue and the factors contributing to improved tolerance are not well known. In this study, the constitutively flocculating Saccharomyces cerevisiae strain CCUG 53310, with good ability to ferment toxic hydrolysates, was compared with S. cerevisiae CBS 8066 in order to characterize the mechanisms of flocculation and the fermentative performance in different inhibitory media. The flocculation of CCUG 53310 depended on cell wall proteins and was partly inhibited by mannose. The flocculating cells also exhibited a significantly higher hydrophobicity than the cells of the non-flocculating strain CBS 8066, which might contribute to the flocculation. The flocculating strain was more tolerant to carboxylic acids and furan aldehydes, but more sensitive to phenolic compounds. Surprisingly, the expression increase of YAP1, ATR1 and FLR1, known to confer resistance against lignocellulose-derived inhibitors, upon addition of various inhibitors to the fermentation medium, was less in CCUG 53310 than in CBS 8066 in most cases. This indicates that the flocculating strain experienced the cultivation conditions as less stressful. The flocculation in itself is a likely cause of this by creating subinhibitory local levels of inhibitors for most cells, allowing the cells in flocs to experience a lower collective stress level.
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